This revised application has the long-term goal of identifying the proteases that are important for the development and progression of cancers of the prostate, breast, and squamous epithelium. As a corollary, we plan to identify their natural substrates and the pathways of activation of proteolytic cascades involved in cancer progression to determine their function in cancer. Project 1 will determine the role of transmembrane serine protease (MT-SP)-I and -2 in cancer progression by developing macromolecular protease inhibitors of their enzymatic function from ecotin or single chain antibody scaffolds. This has already been done for MT-SP1. These antibodies will be used in transplant and transgenic models of prostate cancer. Project 1 will focus also on characterizing the function of a homologue of MT-SP1, MT-SP2, which is expressed primarily in the prostate. Project 2 will focus primarily on cysteine proteases, testing small molecule inhibitors for identifying proteases and protease efficacy in vivo and in experimental therapeutic trials of inhibitors of cysteine, serine and matrix metalloproteases (MMP) in transgenic models of pancreatic islet cell and prostate cancer. Project 3 will examine the temporal and spatial expression and function of metalloproteases and their substrates in transgenic models of neoplastic progression using mice with mutations regulating proteolysis. The focus will be primarily on MMP-2, -3, and -13 and tissue inhibitor of metalloproteinases (TIMP)-I in squamous cell and prostate cancer. The Biochemistry Core will continue to develop new technology for identifying new proteases and to apply these methodologies to tumor specimens from patients with and ovarian prostate cancer in collaboration with Project 1. The Animal Core will breed and maintain transgenic and MMP and MT-SP1 mutant mice, including double knockouts, on inbred backgrounds including RAG1-/-, and will generate a tissue-specific MT-SP1 knockout mouse with Project 1. These will be crossed into transgenic mice prone to squamous cell, pancreatic islet and prostate cancer for all three projects. The Chemistry Core will create a combinatorial library of small molecules in order to develop potent and specific inhibitors of MT-SP1 and of specific MMPs. The molecules will be used to dissect the function of these proteases and to determine whether they inhibit neoplastic transformation, tumor growth, invasion, and/or metastasis, in vitro or in vivo. The PPG will take advantage of the Biomolecular Resource Center and the following UCSF Comprehensive Cancer Center, and Prostate SPORE resources in the proposed research: the Biostatistics, Tissue, Informatics, Gene Expression, Clinical Research Genomic and Transgenic Animal Cores.